Project Summary: Wolbachia are a genus of endosymbiotic bacteria that comprise a promising, cost-effective tool to curb Zika and dengue arboviral transmission based on two key facets. First, Wolbachia block pathogenic RNA viruses by inhibiting their replication in arthropods. Second, Wolbachia selfishly alter sperm and egg via a process termed cytoplasmic incompatibility (CI) that can drive the bacteria into host populations. CI is expressed as embryonic lethality in crosses between infected males and uninfected females, but this lethality is rescued in crosses between infected males and infected females, which are the transmitting sex of Wolbachia. Consequently, CI is deployed in field trials to either suppress mosquito population sizes or replace uninfected populations with infected individuals resistant to arboviral infection. We recently exposed a genetic model of CI wherein expression of two genes (cifA and cifB) causes embryonic lethality when expressed in testes, and expression of one of the same genes (cifA) rescues lethality when expressed in ovaries. Prior to embryonic lethality, several post-fertilization defects arise including delayed breakdown of the paternal nuclear envelope, mitotic arrest, and chromatin bridging. As Wolbachia are stripped from sperm during spermatogenesis, the defects caused by cifA and cifB may be due to uncharacterized pre-fertilization impairments to sperm integrity. Despite four decades of intense research and current applications to vector control efforts, the details surrounding these pre-fertilization impairments remain a central enigma. The overarching hypothesis of the proposed research is cifA and cifB encode proteins that alter sperm integrity to cause CI. In Aim 1, we will use cytochemical, enzymatic, and transgenic assays to determine the types, strength, and genetic bases of sperm modifications imposed by the Cif proteins from wMel Wolbachia released in field trials by the World Mosquito Program. In Aim 2, we will investigate localization patterns of the Cif proteins during spermatogenesis and storage. We will also identify important interactions between the Cif proteins and either host or Wolbachia proteins. Knockout and transgenic experiments will interrogate the necessity of the protein-protein interactions for expression of CI. Finally in Aim 3, we will evaluate if the natural CI proteins or protein complex can be experimentally isolated and successfully injected into uninfected hosts to recapitulate CI and rescue. In these experiments, we will evaluate a novel association between the Cif proteins and bacteriophage WO particles from Wolbachia. Studies have yet to yield a mechanistic breakthrough for the natural CI defects afflicting gametes, and the rising interest in deploying Wolbachia to curb arbovirus transmission necessitates an urgent understanding of the events underpinning the CI drive system. If successful, this research will fundamentally advance studies of CI modifications and inform Wolbachia's ongoing efficacy and delivery as a natural tool to control arthropods worldwide.